The primary transcripts of most eukaryotic structural genes (pre- mRNAs) contain intervening sequences that are removed by RNA splicing. In some instances, alternative splicing of a common pre- mRNA provides an important mechanism for regulating gene expression. We have been studying the biochemical mechanisms involved in pre-mRNA splicing using in vitro systems. Splicing occurs in a large multicomponent complex, which contains both proteins and small nuclear ribonucleoprotein particles (snRNPs). It is likely that once this complex is formed, the splice sites that will ultimately be joined have already been determined. To study the early events involved in splicing complex assembly we will pursue two complementary approaches that have already been initiated in the laboratory. First, we will further purify the proteins involved in pre-mRNA splicing and characterize their mechanisms of action. In addition to proteins, the U1, U2, U5, and U4/U6 snRNPs are splicing factors. We will use methods recently developed in our laboratory for reconstituting all of these snRNPs from their isolated RNA moieties and protein components. This method should allow us to further study the interaction of snRNPs with the pre-mRNA and with each other during splicing complex assembly. In addition, we will initiate a dissection of each snRNP into functional domains by determining the snRNA regions and snRNP structural polypeptides required for the various snRNP activities. Finally, experiments are proposed to determine how splice sites are accurately selected and the mechanisms involved in regulated alternative splicing.